The project objective is to identify and characterize genes involved in bacterial cell differentiation.[unreadable] Vegetative cells of the filamentous cyanobacterium Nostoc punctiforme (strain ATCC 29133) are capable of[unreadable] cellular differentiation into spore-like akinetes that function in perennation. N. punctiforme is an excellent[unreadable] model system in which to study cellular differentiation with respect to its recently sequenced genome, well[unreadable] developed genetic system, and availability of a newly developed DNA microarray. The first specific aim of[unreadable] this proposal is to identify genes differentially expressed during akinete differentiation using microarray[unreadable] analysis. This study will utilize a glucose-6-phosphate dehydrogenase mutant strain of N. punctiforme,[unreadable] designated UCD 466, that differentiates a large proportion of vegetative cells into akinetes following dark[unreadable] incubation in the presence of fructose. The wild-type strain is capable of heterotrophic growth under similar[unreadable] conditions and does not differentiate into akinetes. The DNA microarray technique will be performed with[unreadable] samples from various time points during akinete formation to identify regulated genes. The second specific[unreadable] aim is to construct and physiologically characterize mutants in selected differentially expressed genes by[unreadable] targeted gene replacement. Transcriptional reporter constructs will also be generated for selected genes and[unreadable] introduced into the wild-type and UCD 466 strains to determine temporal and spatial (cell-type specific)[unreadable] expression patterns. Promoter sequences from differentially expressed genes will be determined and the[unreadable] consensus sequence for common promoter elements used for future characterization of associated[unreadable] regulatory proteins. Functional characterization of differentially expressed gene products in akinetes will[unreadable] lead to a better understanding of how this specialized cell type can withstand harsh conditions. Regulatory[unreadable] mutants generated in this work will be used to identify regulons and regulatory cascades involved in akinete[unreadable] differentiation and allow identification of those that overlap differentiation processes in other cell types.